Journal of Fish Diseases 2007, 30, 471–482 Selected pathological, immunohistochemical and ultrastructural changes associated with an infection by Diphyllobothrium dendriticum (Nitzsch, 1824) (Cestoda) plerocercoids in Coregonus lavaretus (L.) (Coregonidae) B S Dezfuli1, F Pironi1, E Simoni1, A P Shinn2 and L Giari1 1 Department of Biology, University of Ferrara, Ferrara, Italy 2 Institute of Aquaculture, University of Stirling, Stirling, UK Abstract The pathological changes induced by an infection of Diphyllobothrium dendriticum (Nitzsch, 1824) plerocercoids in powan, Coregonus lavaretus (L.), from Loch Lomond, Scotland, were assessed using immunohistochemical and ultrastructural techniques. In a sample of 26 powan, the occurrence of encysted plerocercoids of D. dendriticum on the outer surface of the stomach was 38.5% (n = 10) with the number of cysts ranging from 4 to 15 and measuring 4.2  1.0 mm · 3.4  0.9 mm (mean  SD). Histological examination of intestinal samples also revealed plerocercoids (2–21) encapsulated within a proliferation of mesenteric fibrous tissues of the gastric wall and, occasionally, by the gut lamina propria-submucosa and lamina muscularis. In section, cysts were tri-layered and were formed from a series of concentric whorls of fibroblast and collagen fibre-based connective elements. The extent of necrosis within each muscle layer and the serosa of the stomach differed, notably within the latter that was marked by a chronic inflammatory reaction and fibrosis. Within the cyst and around it, a large number of degranulating mast cell/eosinophilic granule cells were seen, in addition to melano-macrophage centres. Immunohistochemical staining of sections of infected stomach revealed a high density of elements, in close proximity to plerocercoids, staining positive for serotonin, bombesin, substance P and galanin.  2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd Correspondence B S Dezfuli, Department of Biology, University of Ferrara, Via Borsari 46, 44100 Ferrara, Italy (e-mail: dzb@unife.it) 471 Uninfected material did not present the same levels of activity. Sections through both infected and uninfected tissue were also tested for elements containing vasoactive intestinal peptide, met-enkephalin, calcitonin gene-related peptide, neuropeptide Y and nitric oxide synthase, but these were absent. Keywords: Coregonus lavaretus, Diphyllobothrium dendriticum, immunohistochemistry, pathology, plerocercoid, stomach. Introduction While Diphyllobothrium Cobbold, 1858 (Cestoda: Pseudophyllidea) infections of man have received much attention (Bonsdorff 1977; Vaiani, Terramocci, Crotti, Gustinelli, Invernizzi, Fioravanti & Pampiglione 2006), they also represent important infestations of wild and cultured freshwater fish (Hoffman & Dunbar 1961; Wootten & Smith 1979; Halvorsen & Andersen 1984). Diphyllobothrium dendriticum (Nitzsch, 1824) uses a copepod as its first host, a planktivorous fish such as those belonging to the Coregonidae, Salmonidae and Gasterosteidae as its second intermediate host and a larid gull as its definitive host (Wright & Curtis 2000). Generally, the most intense reaction to the parasite occurs when the fish acts as an intermediate stage where the metacestode becomes encapsulated within the body cavity or within various body organs. Mortalities of fish linked to infections of Diphyllobothrium have been recorded from North Journal of Fish Diseases 2007, 30, 471–482  2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd America (Becker & Brunson 1967; Bérubé & Curtis 1986), South America (Torres, Franjola, Figueroa, Schlatter, Gonzalez, Contreras & Martin 1981; Revenga 1993; Torres, Lopez, Cubillos, Lobos & Silva 2002), and from Europe (Fraser 1960a,b; Hoffman & Dunbar 1961; Henricson 1977; Halvorsen & Andersen 1984; Rahkonen, Aalto, Koski, Särkkä & Juntunen 1996), and their impact on wild populations of fish, however, is currently unknown (Rahkonen & Valtonen 1997). The pathology associated with infections of Diphyllobothrium spp. has been detailed by a number of authors (Torres et al. 1981, 2002; Rahkonen et al. 1996), while the immune response and tissue reaction of the host has been comprehensively investigated in naturally acquired infections of D. dendriticum and Diphyllobothrium ditremum and following the experimental infection of D. dendriticum in Oncorhynchus mykiss (Walbaum) by Sharp, Pike & Secombes (1989, 1991, 1992). Enteric helminth infections commonly cause inflammation of the host alimentary canal leading to alterations in gastrointestinal function such as enhanced secretion and gut propulsive motility (Palmer & Greenwood-Van Meerveld 2001; Dezfuli, Giari, Arrighi, Domeneghini & Bosi 2003). Intestinal worm infections in mammals, for example, have been shown to induce alterations in the concentration of several neuromodulators in host tissues and plasma (McKay, Halton, Johnston, Shaw, Fairweather & Buchanan 1991; Fairweather 1997; Eysker & Ploeger 2000). A recent plethora of studies in fish have shown that helminths can induce a marked change in the concentration of certain neuromodulators (Dezfuli, Arrighi, Domeneghini & Bosi 2000; Dezfuli, Giari, Simoni, Bosi & Manera 2002a; Dezfuli, Pironi, Giari, Domeneghini & Bosi 2002b; Dezfuli et al. 2003; Bosi, Shinn, Giari, Simoni, Pironi & Dezfuli 2005a; Dezfuli, Giari, Simoni, Shinn, Manera & Bosi 2005). These studies suggest that the presence of a parasite within a host can induce the formation of a network of nervous fibres at the site of inflammation which is demonstrated by the increase in the number of immunoreactive elements [e.g. bombesin, substance P (SP) and galanin] within the newly formed network. Although a similar response was expected for the powan–cestode system, almost nothing was known regarding the nervous system of infected and uninfected fish stomach tissue. The aim of this study was to assess which neuromodulators 472 B S Dezfuli et al. Diphyllobothrium dendriticum in powan were present in normal gut tissue of Coregonus lavaretus and whether the presence of D. dendriticum plerocercoids influenced their distribution and concentration. The work of Sharp et al. (1992), using experimental infections, provided a clear account of the sequential development of Diphyllobothrium and its migration through host tissue. A preliminary study of powan–Diphyllobothrium infected material by the current authors demonstrated a high number of mast cells/eosinophilic granule cells (EGCs) suggesting that they were part of the host defence mechanism as has been shown within other teleosts (Reite 1997, 2005; Reite & Evensen 2006). A high number of such cell types, which are usually active in chronic inflammation (Reite & Evensen 2006), suggested that the parasite is causing damage by inducing degranulation of mast cells/EGCs with the release of inflammatory cytokines. In addition to these cells, melano-macrophage centres (MMCs) were also observed within the tissues forming the cyst around the D. dendriticum plerocercoid. Proliferation of MMCs has been linked to a number of pathological and physiological changes in the host (Vogelbein, Fournie & Overstreet 1987; Wolke 1992; Couillard, Williams, Courtenay & Rawn 1999; Agius & Roberts 2003). The aim of this project was to conduct a detailed study of the infected tissues to elucidate the role of the key cell types (i.e. EGCs and MMCs) in the defence mechanisms of the host and to assess the distribution and a number of a range of immunoreactive elements in uninfected and infected host stomach tissue. Materials and methods The guts of 26 powan, C. lavaretus, (32.03  0.63 cm fork length; 355.96  17.39 g total body weight) were analysed from powan collected in two gill net samples (July 2003 and August 2004) from Loch Lomond (5310¢N, 439.1¢W). The powan were given a lethal dose of MS222 (Sandoz, Basel, Switzerland), weighed and measured before they were dissected ventrally, sexed and the alimentary canals observed in situ. In parasitized fish, the number and position of each encysted plerocercoid on the outer surface of the stomach were recorded; the plerocercoids were removed in situ and fixed in chilled (4 C) BouinÕs fluid for 7 h. The samples were then processed routinely for paraffin embedding, cut in 5-lm-thick sections and stained either with haematoxylin and B S Dezfuli et al. Diphyllobothrium dendriticum in powan Journal of Fish Diseases 2007, 30, 471–482 eosin (H&E), Azan-Mallory, periodic acid–Schiff (PAS), alcian blue/PAS, or used for immunohistochemistry. The latter was done according to the peroxidase–antiperoxidase method detailed in Dezfuli et al. (2002b, 2003). The anti-sera, the working dilution and the incubation times used for each of the neuromodulators are given in Table 1. The controls for the specificity of the immunohistochemical reactions were performed by the preabsorption of each anti-serum with the corresponding antigen (Table 2). The control for the anti-protein gene-product 9.5 (PGP9.5) serum was performed by incubating the sections with rabbit normal serum using the same conditions for the primary anti-serum. Tissue sections taken from a rat and a pig were used as positive controls. Evaluation of the distribution and frequency of the immunoreactive elements were based on subjective estimates after the examination at 20· of five sections of the stomach of 18 powan (10 parasitized and 8 uninfected). Each section was scored depending on whether there was a low (+), medium (++) or a high (+++) occurrence of immunoreactive elements. For light and electron microscopy, infected stomach tissues measuring up to 8 · 8 mm in diameter were fixed for 2 h in a chilled (4 C) 2% glutaraldehyde solution buffered at pH 7.2 with 0.1 m sodium cacodylate. Thereafter, the pieces were rinsed for 12 h with 0.1 m sodium cacodylate buffer containing 6% sucrose. The tissues were then post-fixed in 1% osmium tetroxide in the same buffer for 2 h, dehydrated through a graded ethanol series, transferred to propylene oxide and then embedded in an Epoxy-Araldite mixture (Fluka, Buchs, Switzerland). Semi-thin sections (5 lm) were cut on a Reichert Om U2 ultramicrotome (Reichert-Jung, Vienna, Austria) and stained with methylene blue. Ultra-thin sections (90 nm) were stained with a solution of 4% uranyl acetate in 50% alcohol and ReynoldÕs lead citrate and examined using a Hitachi H-800 electron microscope (Hitachi, Tokyo, Japan). For comparative purposes, the uninfected stomachs of 8 C. lavaretus were also processed. Light photomicrographs were taken using a Nikon microscope ECLIPSE 80i and a Nikon stereomicroscope (Nikon, Tokyo, Japan). Morphometric measurements of key features of the Table 1 The primary anti-sera used in this study Anti-sera raised in rabbit Code Source Dilution Incubation Bombesin Bombesin CGRP Galanin Met-enkephalin Met-enkephalin NOS NPY NPY Serotonin Substance P VIP VIP 1400-0004 IHC 7113 IHC 7181 T-4330 (IHC 7153) IHC 8602 AB 1975 sc-648 6730-0004 IHC 7180 AB 938 T-4107 (IHC 7451) CA-08-340 9535-0204 Biogenesis Ltd., Poole, UK Peninsula Lab., Inc., Belmont, CA, USA Peninsula Lab., Inc., Belmont, CA, USA Peninsula Lab., Inc., Belmont, CA, USA Peninsula Lab., Inc., Belmont, CA, USA Chemicon Int., Temecula, CA, USA Santa Cruz Biot., Santa Cruz, CA, USA Biogenesis Ltd., Poole, UK Peninsula Lab., Inc., Belmont, CA, USA Chemicon Int., Temecula, CA, USA Peninsula Lab., Inc., Belmont, CA, USA Genosys Biotechnologies, Cambridge, UK Biogenesis Ltd., Poole, UK 1:200 1:200 1:400 1:500 1:500 1:500 1:200 1:50 1:500 1:1000 1:500 1:1000 1:50 Overnight at Overnight at 24 h at 4 C Overnight at Overnight at Overnight at Overnight at 24 h at 4 C 24 h at 4 C Overnight at Overnight at Overnight at 24 h at RT 4 C 4 C 4 4 4 4 C C C C 4 C 4 C 4 C CGRP, calcitonin gene-related peptide; NOS, nitric oxide synthase; NPY, neuropeptide tyrosine; VIP, vasoactive intestinal peptide; RT, room temperature. Table 2 Details of the peptides used for the absorption controls  2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd Peptide Code Source Bombesin CGRP Galanin Met-enkephalin NOS NPY Serotonin Substance P VIP B 4272 H 4924 H 1365 H 2785 sc-648 P H 6375 H 9523 H 1890 V 3628 Sigma Chemicals, St. Louis, MO, USA Bachem AG, Bubendorf, Switzerland Bachem AG, Bubendorf, Switzerland Bachem AG, Bubendorf, Switzerland Santa Cruz Biotechnologies, Inc., Santa Cruz, CA, USA Bachem AG, Bubendorf, Switzerland Sigma Chemicals, St. Louis, MO, USA Bachem AG, Bubendorf, Switzerland Sigma Chemicals, St. Louis, MO, USA CGRP, calcitonin gene-related peptide; NOS, nitric oxide synthase; NPY, neuropeptide tyrosine; VIP, vasoactive intestinal peptide. 473 Journal of Fish Diseases 2007, 30, 471–482 plerocercoid-infected tissue were recorded with the aid of light microscopy, using computerized image analyser software (Lucia G 4.8, Laboratory Imaging, Prague, Czech Republic). Results  2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd Figure 1a shows a histological section through the stomach of an uninfected C. lavaretus, illustrating the respective layers of the gastric wall and their integrity. From the 26 C. lavaretus examined, the stomachs of 10 (38.5%) fish were parasitized with plerocercoids of D. dendriticum. Each stomach had between 4 and 15 plerocercoids within a cyst measuring 4.2  1.0 · 3.4  0.9 mm (mean  SD) (Fig. 1b,c). The larger cysts on the external surface of the stomach were evident as loosely attached nodules (Fig. 1b,c), while others were intramural. Sections through the cysts revealed a fully developed, tightly packaged plerocercoid within (Fig. 1c), or occasionally, alongside necrotic tissue (Fig. 1c). Sections through the stomach wall of infected powan revealed a number of plerocercoids (range 2–21 larvae per infected host) distributed at various depths throughout the tissue (Fig. 1d). Cysts were observed to consist of three distinct areas, an inner fibrous area where necrotic cells were found in close proximity to the plerocercoid (Fig. 2a,b), a middle region consisting of degenerating epithelioid cells which give way to giant cells towards the outer zone of the cyst (Fig. 2b,c) and, finally, the outer layer made up largely of concentrically oriented fibroblasts and collagen (Fig. 2e). Larvae appear to penetrate the stomach wall and migrate to the serosa (Fig. 1d,e) and as they migrate and grow, they elicit a chronic inflammatory response, which results in fibrosis and larval encapsulation (Figs 1d & 2a). By the time most plerocercoids reach the serosa, they have attained their maximum size and remain attached to the serosal surface as a loose nodule (Fig. 1b,c) or as a protuberance within the gastric wall (Fig. 1b,e). As mentioned above, intramural plerocercoids were also found within the gastric wall. In addition to encapsulation of the parasite, a range of other host responses were evident including the presence of MMC in close proximity to the plerocercoid (Fig. 1f). Also, within the cyst wall and in the fibrous tissues surrounding the cyst, a large number of EGCs were observed (Fig. 2a,b). In several cases where the encysted plerocercoid was found firmly attached to the outer surface of the 474 B S Dezfuli et al. Diphyllobothrium dendriticum in powan stomach, a section through the cyst revealed a large number of EGCs in a configuration suggesting that they were passing through the muscle layer and migrating towards the plerocercoid (Fig. 2a,c). An electron microscopic examination of the EGCs scattered among the collagen fibres and fibroblasts (Fig. 2d,e) revealed that, ultrastructurally, they have a large number of granules which were frequently depleted suggesting degranulation (Fig. 2f). These cells, typically, had an undifferentiated cytoplasm, lacked organelles and possessed nuclei that appeared irregular in shape and were often pyknotic (Fig. 2f). In the present study, the prevalence of immunoreactive elements responding to 13 different antisera were assessed on histological sections cut from both uninfected and D. dendriticum-infected stomachs of C. lavaretus. Although both the uninfected and infected stomachs were positive for four different neuromodulators, the most marked difference between the two sets of samples was the number of positive cells within the lamina propriasubmucosa (Fig. 3a) and the muscle layers of the infected stomach. Indeed, in parasitized stomachs high numbers of cells positive to serotonin were seen around the plerocercoid (Fig. 3b,c; Table 3). The uninfected stomachs had a low number of immunoreactive elements positive for serotonin, bombesin, SP and galanin (Table 3). In infected stomachs, we found a high number of bombesinpositive nerve fibres principally on the surface of the cyst as well as bombesin-positive endocrine epithelial cells within the mucosa (Fig. 3d,e,f; Table 3). Similarly, numerous nerve fibres running throughout the serosa (Fig. 3h) of infected tissue and in the tissues in close proximity to the plerocercoid were positive for galanin (Fig. 3i; Table 3). Finally, the use of the SP anti-sera also indicated a large number of positive elements in the muscle layers of the stomach, within the cyst capsule and in the tissues around it (Fig. 3g, Table 3). Histochemical staining of stomach sections taken from both infected and uninfected hosts with the remaining anti-sera anti-neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), met-enkephalin, calciotonin gene-related peptide (CGRP) and nitric oxide synthase (NOS), did not reveal any positive immunoreactive structures. Discussion While many studies have dealt with tissue responses to infections with plerocercoids of Diphyllobothrium Journal of Fish Diseases 2007, 30, 471–482 B S Dezfuli et al. Diphyllobothrium dendriticum in powan Figure 1 (a) A stereograph image of a section through the stomach of uninfected Coregonus lavaretus: note the integrity of the gastric wall (bar = 100 mm). (b) A stereograph image of two large encysted plerocercoids (arrow heads) of Diphyllobothrium dendriticum on peritoneal surface of the stomach, the larvae are loosely attached to the organ; one intramural larva (arrow) is visible (bar = 150 mm). (c) Section of infected open stomach, with a cyst apparently free in the peritoneum. Note the size of the cyst and the space occupied by the fully developed plerocercoid: arrow shows necrotized tissue (stereograph image, bar = 120 lm). (d) Plerocercoids (arrows) in different layers of the gastric wall. Host reaction around the cyst is appreciable (bar = 250 lm). (e) A developed plerocercoid within a ÔtunnelÕ migrates toward the peritoneum: near the larval body, presence of necrotic tissue (arrows) is evident (bar = 250 lm). (f) Within the thickness of the cyst and near the parasite body (asterisk) macrophage aggregates (MAs) (arrows) are visible (bar = 50 lm).  2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd 475 B S Dezfuli et al. Diphyllobothrium dendriticum in powan Journal of Fish Diseases 2007, 30, 471–482 (a) (d) (b) (c) (e) (f) Figure 2 (a) Semithin sections from an infected stomach with a cyst, arrows show the migration of eosinophilic granule cells (EGCs) through the muscle layer (M) toward the plerocercoid (asterisk), white arrow points towards the lumen side of the stomach (bar = 100 lm). (b) Larval body (asterisk) and the cyst wall; arrows indicate two EGCs (bar = 10 lm). (c) EGCs (arrows) within fibrous tissue surrounding the cyst (bar = 10 lm). (d) EM of three EGCs (arrow heads) within connective tissue (arrows) (bar = 4.80 lm). (e) EM of several EGCs (arrow heads) within collagen fibres (arrows) (bar = 4.85 lm). (f) High magnification of a mast cell/EGC: note degranulation (arrows) and pyknotic nucleus (asterisk) (bar = 1.33 lm).  2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd in different species of fish (Halvorsen 1970; Gonzalez, Torres, Figueroa, Contreras & Franjola 1978; Otto & Heckmann 1984; OÕNeill, White, Sims & Barber 1988; Weiland & Meyers 1989; Torres et al. 2002), the work of Sharp et al. (1992) provided clear details of the sequential development of the immune response and cyst development in O. mykiss experimentally infected with D. dendriticum. From the study of Sharp et al. (1992) and the current study, it would appear that, ultrastructurally, the cyst encapsulating the plerocercoids possesses the same features as those produced in response to other endohelminths such as the acanthocolpid digenean Stephanochasmus baccatus 476 known to parasitise at least four species of flatfish (Sommerville 1981) and Rhipidocotyle johnstonei in Pleuronectes platessa (L.) (Pulsford & Matthews 1984). In the current study, a high number of EGCs were encountered within the walls of the parasite cyst and in the immediate tissues surrounding it. These cells increased in number towards the plerocercoid encysted on the outer surface of the stomach, possibly as a consequence of their migration through the stomach wall towards the site of parasite infection. EGCs are particularly numerous within the gut tissue of salmonids where they form the discrete stratum granulosum (Yasutake & Wales B S Dezfuli et al. Diphyllobothrium dendriticum in powan Journal of Fish Diseases 2007, 30, 471–482  2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd (a) (b) (c) (d) (e) (f) (g) (h) (i) Figure 3 (a) Large number of structures (arrows) immunoreactive to serotonin in the tunica propria-submucosa and positive nerve fibres (arrowheads) within the muscle layer of infected stomach of Coregonus lavaretus (bar = 100 lm). (b) Numerous elements (arrows) positive to serotonin around a cyst (asterisk) (bar = 50 lm). (c) Cells (arrows) immunoreactive to serotonin encircle fully developed plerocercoid (asterisk) (bar = 50 lm). (d) Endocrine cells (arrows) containing bombesin in the gastric epithelium of infected powan (bar = 30 lm). (e) Nerve fibres (arrow) on surface of the cyst (bar = 100 lm). (f) Higher magnification of previous micrograph, note numerous bombesin-positive fibres (arrows) (bar = 30 lm). (g) Large number of elements (arrows) reactive to substance P in the muscular layer and in the connective inflammatory tissue around the parasite (asterisk) (bar = 100 lm). (h) Galanin-positive nerve fibres (arrowheads) near the connective tissue surrounding a plerocercoid (asterisk) beneath the serosa (arrows) (bar = 100 lm). (i) Nerve fibres (arrows) immunoreactive to galanin near the parasite body (asterisk) (bar = 50 lm). 477 Journal of Fish Diseases 2007, 30, 471–482 Table 3 The response of uninfected and Diphyllobothrium dendricticum-infected stomach sections to a range of primary anti-sera Anti-sera Uninfected stomach Infected stomach Bombesin Galanin Serotonin Substance P ++ + + + +++ +++ +++ +++ The table shows a score of the immunoreactive elements found for each anti-sera in each tissue type. Key: +: low presence, ++: medium presence, +++: high presence of structures immunoreactive to the specified anti-serum.  2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd 1983). They have also been found in high numbers in Salmo trutta (L.) infected with the cestode Cyathocephalus truncatus (Dezfuli et al. 2000) and in 3-spined sticklebacks, Gasterosteus aculeatus (L.), parasitized with the microsporean Glugea anomala (Dezfuli, Giari, Simoni, Shinn & Bosi 2004). EGCs are known to have an immune function similar to that of mammalian mast cells and their degranulation is in response to acute tissue damage due to pathogens/parasites (Reite 1998, 2005); chronic inflammatory reactions in gills or intestinal tissues induce a local increase in EGC numbers (Reite 1998). Aggregations of melano-macrophages were also recorded in close proximity to the encysted parasite. Increases in MMCs have been associated with a range of physiological and pathological factors including ageing, starvation, the presence of infectious disease/pathogens and intoxication (Vogelbein et al. 1987; Wolke 1992; Couillard & Hodson 1996; Couillard et al. 1999; Agius & Roberts 2003). Their response to parasitic infection was comprehensively demonstrated by Vogelbein et al. (1987) following the experimental infection of Rivulus marmoratus Poey with the protozoan Calyptospora funduli (Duszynski, Solangi & Overstreet 1979; Overstreet, Hawkins & Fournie 1984). Thirty days after infection, multifocal granulomatous lesions were noticed within the liver, followed by a progressive increase in melanin and lipofucsin within the resulting MMCs 50–150 days postinfection. The main detrimental effects of most endoparasitic helminths are localised at the site of infection (Hoste 2001) where, for example, worms induce structural changes to the digestive system, which might include local neuroendocrine structures with resulting alterations in the functions of the gastrointestinal tract (Castro 1992; Fairweather 1997; Fox 478 B S Dezfuli et al. Diphyllobothrium dendriticum in powan 1997; Palmer & Greenwood-Van Meerveld 2001). Most of the studies on the above alterations focused on parasitic infections in mammals (Fox 1997; Eysker & Ploeger 2000; Mercer, Mitchell, Moar, Bissett, Geissler, Bruce & Chappell 2000), although there is a growing number of parallel studies in fish (see Dezfuli et al. 2000, 2002b, 2003, 2004, 2005; Bosi, Di Giancamillo, Arrighi & Domeneghini 2004a; Bosi et al. 2005a; Bosi, Domeneghini, Arrighi, Giari, Simoni & Dezfuli 2005b; Bermúdez, Vigliano, Quiroga, Nieto, Bosi & Domeneghini 2006). Our immunohistochemical analysis showed an increase in the immunoreactive elements responding to the serotonin, bombesin, SP and galanin anti-sera in stomach sections taken from D. dendriticum-infected powan. Of these, cells that were immunoreactive to the serotonin (5-HT) antiserum were found in the lamina propria-submucosa and the muscle layers of the stomach. Information on this neuromodulator and its role in the immune system is scarce (Khan & Deschaux 1997), but its presence has been documented in other fish– parasite systems including S. trutta infected with another cestode, Cyathocephalus truncatus (Dezfuli et al. 2000), an acanthocephalan Pomphorhynchus laevis (Dezfuli et al. 2003), a microsporean Glugea anomala (Dezfuli et al. 2004) and also in the hearts of powan infected with the digenean Ichthyocotylurus erraticus (Dezfuli et al. 2005). A significant increase in serotonin activity in the intestines and muscles of rats infected with Trichinella spiralis and T. pseudospiralis has also been reported (Terenina, Asatrian & Movsessian 1997). It has been suggested that this biogenic amine affects vascular permeability and lymphocyte function (Lee, Swieter & Befus 1986), exerting a variety of effects that may favourably affect parasite survival (Fairweather 1997). If parallels between these host–parasite systems can be drawn, this suggests that an infection of D. dendriticum induces the recruitment of cells to secrete serotonin at the site of infection to ensure their survival. Elements that were immunoreactive to the bombesin anti-serum were found primarily in the gastric epithelium of infected powan and in the nerve fibres of the tissues forming and surrounding the cyst. Bombesin-positive nerve fibres were also reported by Dezfuli et al. (2000, 2003, 2004) within the intestinal folds of S. trutta parasitized with C. truncatus and P. laevis and in Gasterosteus aculeatus infected with Glugea anomala. These findings suggest that bombesin at the site of tissue Journal of Fish Diseases 2007, 30, 471–482  2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd inflammation in fish may act as a putative neurotransmitter in the neo-formed network of nervous fibres. In mammals, bombesin is known to regulate ion transport in the small and large intestine (Kachur, Miller, Field & Rivier 1982; Brown & OÕGrady 1997), but more research is needed to determine the precise role of bombesin and its role in uninfected and parasite infected fish. In mammals, the neuropeptide anti-SP is involved in several neurogenic inflammatory responses such as vasodilatation and plasma extravasation (Abrahamian, Fodor, Gorcs, Galoyan & Palkovits 1991; Onuhoa, Alpar, Chukwulobelu & Nicholls 1999). The work of Sharkey (1992) suggests that the action of SP is not only on the vasculature but also on the mast cells from which histamine and other soluble mediators are released, contributing further to the local inflammatory response. Anti-SP has also been demonstrated in elasmobranchs (Waugh, Wang, Hazon, Balment & Conlon 1993) and teleosts (Davies, Donald & Campbell 1994; Holmgren, Fritsche, Karila, Gibbins, Axelsson, Franklin, Grigg & Nilsson 1994; Waugh, Groff, Platzack, Youson, Olson & Conlon 1995). In the current study, it was observed in the nerve fibres of infected stomach muscle of C. lavaretus and in the network of subtle nerve fibres of the fibrous tissues encapsulating the plerocercoids. Although the function of SP within the cyst is currently unknown, the distribution of immunoreactive elements determined in this study suggests that it serves to stimulate blood flow through the vascular system of the developing cyst. Dezfuli et al. (2002b) also found this neuropeptide in the connective tissue around the gut of a brown trout, S. trutta, infected with Pomphorhynchus laevis. Galanin has been implicated, in fish, to be involved in the olfactory and taste functions, in central visual processing, in somatosensory transmission, in osmoregulation, in sex-specific behaviour and in affecting the cardiovascular system (Cornbrooks & Parsons 1991; Holmqvist & Carlberg 1992; Le Mevel, Mabin, Hanley & Conlon 1998). Although most studies on galanin have focused on its role within the nervous system of vertebrates, it has also recently been reported in the neuroendocrine system of several fish species, both infected and uninfected (see Dezfuli et al. 2004; Bosi et al. 2004a; Bosi, Shinn, Giari, Arrighi & Domeneghini 2004b; Bosi et al. 2005b). 479 B S Dezfuli et al. Diphyllobothrium dendriticum in powan Although the D. dendriticum-infected and noninfected stomachs were negative for a number of other anti-sera (VIP, met-enkephalin, CGRP, NPY and NOS), their presence has been shown by Elbal, Lozano & Agulleiro (1988), and Bosi et al. (2004a,b) to be species-specific. Acknowledgements The authors would like to thank Dr M. Manera from the University of Teramo and Dr G. Bosi from the University of Milan, Italy for providing some useful comments on the preparation of this work. In addition, we would also like to thank Dr Colin Adams, Stuart Wilson and Davy Fettes at the Glasgow University Field Station at Rowardennan for their valuable assistance in the collection of powan from Loch Lomond. This investigation was supported by grants from the Italian Ministry of the University and Scientific Research. References Abrahamian S., Fodor M., Gorcs T., Galoyan A. & Palkovits M. (1991) Neuropeptides in atrial subepicardial ganglia of rats. Acta Morphologica Hungarica 39, 267–278. Agius C. & Roberts R.J. (2003) Melano-macrophage centres and their role in fish pathology. Journal of Fish Diseases 26, 499–509. Becker C.D. & Brunson W.D. (1967) Diphyllobothrium (Cestoda) infection in salmonids from three Washington lakes. Journal of Wildlife Management 31, 813–824. Bermúdez R., Vigliano F., Quiroga M.I., Nieto J.M., Bosi G. & Domeneghini C. (2007) Immunohistochemical study on the neuroendocrine system of the digestive tract of turbot, Scophthalmus maximus (L.), infected by Enteromyxum scophthalmi (Myxozoa). Fish and Shellfish Immunology 22, 252–263. Bérubé M. & Curtis M.A. (1986) Transmission of Diphyllobothrium ditremum to Arctic char (Salvelinus alpinus) in two subarctic Quebec lakes. Canadian Journal of Fisheries and Aquatic Sciences 43, 1626–1634. Bonsdorff B. (1977) Diphyllobothriasis in Man. Academic Press, London. Bosi G., Di Giancamillo A., Arrighi S. & Domeneghini C. (2004a) An immunohistochemical study on the neuroendocrine system in the alimentary canal of the brown trout, Salmo trutta, L., 1758. General and Comparative Endocrinology 138, 166–181. Bosi G., Shinn A.P., Giari L., Arrighi S. & Domeneghini C. (2004b) The presence of a galanin-like peptide in the gut neuroendocrine system of Lampetra fluviatilis and Acipenser transmontanus: an immunohistochemical study. Tissue and Cell 36, 283–292. Bosi G., Shinn A.P., Giari L., Simoni E., Pironi F. & Dezfuli B.S. (2005a) Histopathology and alteration to neuromodulators of Journal of Fish Diseases 2007, 30, 471–482 the diffuse endocrine system of the alimentary canal of farmed Oncorhynchus mykiss (Walbaum) naturally infected with Eubothrium crassum (Cestoda). Journal of Fish Diseases 28, 703–711. Bosi G., Domeneghini C., Arrighi S., Giari L., Simoni E. & Dezfuli B.S. (2005b) Response of the gut neuroendocrine system of Leuciscus cephalus (L., 1758) to the presence of Pomphorhynchus laevis Müller, 1776 (Acanthocephala). Histology and Histopathology 20, 509–518. Brown D.R. & OÕGrady S.M. (1997) Regulation of ion transport in the porcine intestinal tract by enteric neurotransmitters and hormones. Comparative Biochemistry and Physiology 118A, 309–317. Castro G.A. (1992) Intestinal physiology in the parasitized host: integration, disintegration, and reconstruction of systems. Annals of the New York Academy of Sciences 664, 369–379. Cornbrooks E.B. & Parsons R.L. (1991) Sexually dimorphic distribution of a galanin-like peptide in the central nervous system of the teleost fish Poecilia latipinna. Journal of Comparative Neurology 304, 639–657. Couillard C.M. & Hodson P.V. (1996) Pigmented macrophage aggregates: a toxic response in fish exposed to bleached-Kraft mill effluent? Environmental Toxicology and Chemistry 15, 1844–1854. Couillard C.M., Williams P.J., Courtenay S.C. & Rawn G.P. (1999) Histopathological evaluation of Atlantic tomcod (Microgadus tomcod) collected at estuarine sites receiving pulp and paper mill effluent. Aquatic Toxicology 44, 263–278. Davies P.J., Donald J.A. & Campbell G. (1994) The distribution and colocalization of neuropeptides in fish cardiac neurons. Journal of Autonomic Nervous System 46, 261–272. Dezfuli B.S., Arrighi S., Domeneghini C. & Bosi G. (2000) Immunohistochemical detection of neuromodulators in the intestine of Salmo trutta L. naturally infected with Cyathocephalus truncatus Pallas (Cestoda). Journal of Fish Diseases 23, 265–273. Dezfuli B.S., Giari L., Simoni E., Bosi G. & Manera M. (2002a) Histopathology, immunohistochemistry and ultrastructure of the intestine of Leuciscus cephalus (L.) naturally infected with Pomphorhynchus laevis (Acanthocephala). Journal of Fish Diseases 25, 7–14. Dezfuli B.S., Pironi F., Giari L., Domeneghini C. & Bosi G. (2002b) The effect of Pomphorhynchus laevis (Acanthocephala) on occurrence and distribution of putative neuromodulators in the intestine of naturally infected Salmo trutta (L.). Diseases of Aquatic Organisms 51, 27–35. Dezfuli B.S., Giari L., Arrighi S., Domeneghini C. & Bosi G. (2003) Influence of enteric helminths on the distribution of the intestinal endocrine cells belonging to the diffuse endocrine system in brown trout, Salmo trutta L. Journal of Fish Diseases 26, 155–166. Dezfuli B.S., Giari L., Simoni E., Shinn A.P. & Bosi G. (2004) Immunohistochemistry, histopathology and ultrastructure of Gasterosteus aculeatus (L.) tissues infected with Glugea anomala (Moniez 1887). Diseases of Aquatic Organisms 58, 193–202.  2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd Dezfuli B.S., Giari L., Simoni E., Shinn A.P., Manera M. & Bosi G. (2005) Histopathology, ultrastructure and 480 B S Dezfuli et al. Diphyllobothrium dendriticum in powan immunohistochemistry of Coregonus lavaretus hearts naturally infected with Ichthyocotylurus erraticus (Trematoda). Diseases of Aquatic Organisms 66, 245–254. Duszynski D.W., Solangi M.A. & Overstreet R.M. (1979) An unusual new coccidium (Protozoa: Eimeriidae) from the liver of the Gulf killifish (Fundulus grandis). Journal of Wildlife Diseases 15, 543–552. Elbal M.T., Lozano M.T. & Agulleiro B. (1988) The endocrine cells in the gut of Mugil saliens Risso, 1810 (Teleostei): an immunohistochemical and ultrastructural study. General and Comparative Endocrinology 70, 231–246. Eysker M. & Ploeger H.W. (2000) Value of present diagnostic methods for gastrointestinal nematode infections in ruminants. Parasitology 120, S109–S119. Fairweather I. (1997) Peptides: an emerging force in host responses to parasitism. In: Parasites and Pathogens: Effects on Host Hormones and Behavior (ed. by N.E. Beckage), pp. 113–139. Chapman & Hall, International Thomson Publishing, New York. Fox M.T. (1997) Pathophysiology of infection with gastrointestinal nematodes in domestic ruminants: recent developments. Veterinary Parasitology 72, 285– 308. Fraser P.G. (1960a) The occurrence of Diphyllobothrium in trout, with special reference to an outbreak in the West of England. Journal of Helminthology 34, 59–72. Fraser P.G. (1960b) On Diphyllobothrium medium (Fahmy, 1954) parasitic in trout in Great Britain. Journal of Helminthology 34, 193–204. Gonzalez H., Torres P., Figueroa L., Contreras B. & Franjola R. (1978) Researches on Pseudophyllidea (Carus, 1813) in the South of Chile. II. Hepatic and splenic pathology by plerocercoid infections of Diphyllobothrium sp. in Salmo gairdneri Richardson, 1836 of Calafquen Lake. Indian Journal of Parasitology 2, 127–129. Halvorsen O. (1970) Studies on the helminth fauna of Norway. XV. On the taxonomy and biology of plerocercoids of Diphyllobothrium Cobbold, 1958 (Cestoda, Pseudophyllidea) from north-western Europe. Nytt Magasin for Zoology 18, 113–174. Halvorsen O. & Andersen K. (1984) The ecological interaction between Arctic charr, Salvelinus alpinus (L.), and the plerocercoid stage of Diphyllobothrium ditremum. Journal of Fish Biology 25, 305–316. Henricson J. (1977) The abundance and distribution of Diphyllobothrium dendriticum (Nitzsch) and D. ditremum (Creplin) in the char Salvelinus alpinus (L.) in Sweden. Journal of Fish Biology 11, 231–248. Hoffman G.L. & Dunbar C.E. (1961) Mortality of eastern brook trout caused by plerocercoids (Cestoda: Pseudophyllidea) in the heart and viscera. Journal of Parasitology 47, 399–400. Holmgren S., Fritsche R., Karila P., Gibbins I., Axelsson M., Franklin C., Grigg G. & Nilsson S. (1994) Neuropeptides in the Australian lungfish Neoceratodes forsteri: effects in vivo and presence in autonomic nerves. American Journal of Physiology Regulatory Integrative and Comparative Physiology 266, R1568–1577. Journal of Fish Diseases 2007, 30, 471–482 Holmqvist B.I. & Carlberg M. (1992) Galanin receptors in the brain of a teleost: autoradiographic distribution of binding sites in the Atlantic salmon. Journal of Comparative Neurology 326, 44–60. Rahkonen R., Aalto J., Koski P., Särkkä J. & Juntunen K. (1996) Cestode larvae Diphyllobothrium dendriticum as a cause of a heart disease leading to mortality in hatchery-reared sea trout and brown trout. Diseases of Aquatic Organisms 25, 15–22. Hoste H. (2001) Adaptive physiological processes in the host during gastrointestinal parasitism. International Journal for Parasitology 31, 231–244. Reite O.B. (1997) Mast cells/eosinophilic granule cells of salmonids: staining properties and responses to noxious agents. Fish and Shellfish Immunology 7, 567–584. Kachur J.F., Miller R.J., Field M. & Rivier J. (1982) Neurohumoral control of ileal electrolyte transport: I. Bombesin and related peptides. Journal of Pharmacology and Experimental Therapeutics 220, 449–455. Reite O.B. (1998) Mast cells/eosinophilic granule cells of teleostean fish: a review focusing on staining properties and functional responses. Fish and Shellfish Immunology 8, 489–513. Khan N. & Deschaux P. (1997) Role of serotonin in fish immunomodulation. Journal of Experimental Biology 200, 1833–1838. Le Mevel J.C., Mabin D., Hanley A.M. & Conlon J.M. (1998) Contrasting cardiovascular effects following central and peripheral injections of trout galanin in trout. American Journal of Physiology 275, R1118–R1126. Lee T.D.G., Swieter M. & Befus A.D. (1986) Mast cell responses to helminth infection. Parasitology Today 2, 186–191. McKay D.M., Halton D.W., Johnston C.F., Shaw C., Fairweather I. & Buchanan K.D. (1991) Hymenolepis diminuta: changes in the levels of certain intestinal regulatory peptides in infected C57 mice. Experimental Parasitology 73, 15–26. Mercer J.G., Mitchell P.I., Moar K.M., Bissett A., Geissler S., Bruce K. & Chappell L.H. (2000) Anorexia in rats infected with the nematode, Nippostrongylus brasiliensis: experimental manipulations. Parasitology 120, 641–647. OÕNeill J.G., White M.G., Sims T.A. & Barber D.L. (1988) An inflammatory response of the Antarctic silverfish, Pleuragramma antarcticum Boulenger 1902 (Teleostei: Notothenioidei), to infestation by the plerocercoid of a pseudophyllidean cestode (Diphyllobothrium sp.). British Antarctic Survey Bulletin 79, 51–63. Onuhoa G.N., Alpar E.K., Chukwulobelu R. & Nicholls D.P. (1999) Distributions of VIP, substance P, neurokinin A and neurotensin in rat heart: an immunocytochemical study. Neuropeptides 33, 19–25. Otto T.N. & Heckmann R.A. (1984) Host tissue response for trout infected with Diphyllobothrium cordiceps larvae. Great Basin Naturalist 44, 125–132. Overstreet R.M., Hawkins W.E. & Fournie J.W. (1984) The coccidian genus Calyptospora n.sp. and the family Calyptosporidae n. fam. (Apicomplexa), with members infecting primarily fishes. Journal of Protozoology 31, 332–339. Palmer J.M. & Greenwood-Van Meerveld B. (2001) Integrative neuroimmunomodulation of gastrointestinal function during enteric parasitism. Journal of Parasitology 87, 483–504. Pulsford A. & Matthews R.A. (1984) An ultrastructural study of the cellular response of the plaice, Pleuronectes platessa L., to Rhipidocotyle johnstonei nom. nov. (pro-Gasterostomum sp. Johnstone, 1905) Matthews, 1968 (Digenea: Bucephalidae). Journal of Fish Diseases 7, 3–14. Rahkonen R. & Valtonen E.T. (1997) Infection of brown trout with Diphyllobothrium dendriticum procercoids. International Journal for Parasitology 27, 1315–1318.  2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd B S Dezfuli et al. Diphyllobothrium dendriticum in powan 481 Reite O.B. (2005) The rodlet cells of teleostean fish: their potential role in host defence in relation to the role of mast cells/eosinophilic granule cells. Fish and Shellfish Immunology 19, 253–267. Reite O.B. & Evensen Ø. (2006) Inflammatory cells of teleostean fish: a review focusing on mast cells/eosinophilic granule cells and rodlet cells. Fish and Shellfish Immunology 20, 192–208. Revenga J. (1993) Diphyllobothrium dendriticum and Diphyllobothrium latum in fishes from southern Argentina: association, abundance, distribution, pathological effects, and risk of human infection. Journal of Parasitology 79, 379–383. Sharkey K.A. (1992) Substance P and calcitonin gene-related peptide (CGRP) in gastrointestinal inflammation. Annals of the New York Academy of Sciences 664, 425–442. Sharp G.J.E., Pike A.W. & Secombes C.J. (1989) The immune response of wild rainbow trout, Salmo gairdneri Richardson, to naturally acquired plerocercoid infections of Diphyllobothrium dendriticum (Nitzsch, 1824) and D. ditremum (Creplin, 1825). Journal of Fish Biology 35, 781–794. Sharp G.J.E., Pike A.W. & Secombes C.J. (1991) Leucocyte migration in rainbow trout (Oncorhynchus mykiss [Walbaum]): optimization of migration conditions and responses to host and pathogen (Diphyllobothrium dendriticum [Nitzsch]) derived chemoattractants. Developmental and Comparative Immunology 15, 295–305. Sharp G.J.E., Pike A.W. & Secombes C.J. (1992) Sequential development of the immune response in rainbow trout [Oncorhynchus mykiss (Walbaum, 1792)] to experimental plerocercoid infections of Diphyllobothrium dendriticum (Nitzsch, 1824). Parasitology 104, 169–178. Sommerville C. (1981) A comparative study of the tissue response to invasion and encystment by Stephanochasmus baccatus (Nicholl, 1907) (Digenea: Acanthocolpidae) in four species of flatfish. Journal of Fish Diseases 4, 53–68. Terenina N.B., Asatrian A.M. & Movsessian S.O. (1997) Neurochemical changes in rats infected with Trichinella spiralis and T. pseudospiralis. Doklady Biology Science 355, 412–413. Torres P., Franjola R., Figueroa L., Schlatter R., Gonzalez B., Contreras B. & Martin R. (1981) Research on Pseudophyllidea (Carus, 1813) in the South of Chile. IV. Occurrence of Diphyllobothrium dendriticum (Nitzch). Journal of Helminthology 55, 173–187. Torres P., Lopez J.C., Cubillos V., Lobos C. & Silva R. (2002) Visceral diphyllobothriosis in a cultured rainbow trout, Oncorhynchus mykiss (Walbaum), in Chile. Journal of Fish Diseases 25, 375–379. Journal of Fish Diseases 2007, 30, 471–482 B S Dezfuli et al. Diphyllobothrium dendriticum in powan Vaiani R., Terramocci R., Crotti D., Gustinelli A., Invernizzi S., Fioravanti M.L. & Pampiglione S. (2006) Diphyllobothriasis in Como Lake, northern Italy: an update. Parassitologia 48, 297. Oncorhynchus kisutch. Diseases of Aquatic Organisms 6, 175–178. Wolke R.E. (1992) Piscine macrophage aggregates: a review. Annual Review of Fish Diseases 2, 91–108. Vogelbein W.K., Fournie J.W. & Overstreet R.M. (1987) Sequential development and morphology of experimentally induced hepatic melano-macrophage centres in Rivulus marmoratus. Journal of Fish Biology 31, 145–153. Wootten R. & Smith J.W. (1979) The occurrence of plerocercoids of Diphyllobothrium spp. in wild and cultured salmonids from the Loch Awe area. Scottish Fisheries Research Report No. 13. Waugh D., Wang Y., Hazon N., Balment R.J. & Conlon J.M. (1993) Primary structures and biological activities of substance- P-related peptides from the brain of the dogfish, Scyliorhinus canicula. European Journal of Biochemistry 214, 469–474. Wright M.E. & Curtis M.A. (2000) Temperature effects on embryonic development and life cycle of Diphyllobothrium dendriticum. International Journal for Parasitology 30, 849–852. Waugh D., Groff K.E., Platzack B., Youson J.H., Olson K.R. & Conlon J.M. (1995) Isolation, localization and cardiovascular activity of tachykinins from the stomach of the bowfin Amia calva. American Journal of Physiology - Regulatory Integrative and Comparative Physiology 269, R565–R571. Weiland K.A. & Meyers T.R. (1989) Histopathology of Diphyllobothrium ditremum plerocercoids in coho salmon  2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd 482 Yasutake W.T. & Wales J.H. (1983) Microscopic anatomy of salmonids: an atlas. U.S. Department of the Interior. Fish and Wildlife Series Resource Publication 150. Received: 17 November 2006 Revision received: 26 January 2007 Accepted: 5 February 2007